The present invention relates to calcium channel compositions and methods of making and using same. In particular, the invention relates to calcium channel alpha2delta (xcex12xcex4) subunits and nucleic acid sequences encoding them. These compositions are useful in methods for identifying compounds that modulate the activity of calcium channels and for identifying compounds as therapeutic for disease.
Calcium channels are present in various tissues, have a central role in regulating intracellular calcium ion concentrations, and are implicated in several vital processes in animals (e.g., neurotransmitter release, muscle contraction, pacemaker activity, secretion of hormones and other substances, etc.). Thus, changes in calcium influx into cells which are mediated through calcium channels have been implicated in various human diseases such as disorders of the central nervous system and cardiovascular disease.
For example, changes to calcium influx into neuronal cells may be implicated in conditions such as epilepsy, stroke, brain trauma, Alzheimer""s disease, multiinfarct dementia, other classes of dementia, Korsakoff""s disease, neuropathy caused by a viral infection of the brain or spinal cord (e.g., human immunodeficiency viruses, etc.), amyotrophic lateral sclerosis, convulsions, seizures, Huntington""s disease, amnesia, or damage to the nervous system resulting from reduced oxygen supply, poison or other toxic substances (See e.g., Goldin et al., U.S. Pat. No. 5,312,928).
Additionally, changes to calcium influx into cardiovascular cells may be implicated in conditions such as cardiac arrhythmia, angina pectoris, hypoxic damage to the cardiovascular system, ischemic damage to the cardiovascular system, myocardial infarction, and congestive heart failure (Goldin et al., supra). Other pathological conditions associated with elevated intracellular free calcium levels include muscular dystrophy and hypertension (Steinhardt et al., U.S. Pat. No. 5,559,004). While there has been limited success in expressing DNA encoding rabbit and rat calcium channel subunits, little is known about human calcium channel structure, function and gene expression. Additionally, there is limited knowledge in the art of the role of calcium channel types in cell growth control and abnormalities of calcium channels leading to cancer development.
In addition to the implication of calcium channels in animal (including human) diseases, a number of compounds which are currently used for treating various cardiovascular diseases in animals (including humans) are believed to exert their beneficial effects by modulating the functions of voltage-dependent calcium channels present in cells, such as cardiac cells and vascular smooth muscle cells. Nonetheless, there is a paucity of understanding of the pharmacology of compounds which interact with calcium channels. This paucity of understanding, together with the limited knowledge in the art of the human calcium channel types, the molecular nature of the human calcium channel subtypes, and the limited availability of pure preparations of specific calcium channel subtypes to use for evaluating the efficacy of calcium channel-modulating compounds has hampered the rational testing and screening of compounds that interact with the specific subtypes of human calcium channels to have desired therapeutic effects.
The invention provides calcium channel alpha2delta (xcex12xcex4) subunits, as well as amino and nucleic acids encoding them. In one embodiment, the invention provides a substantially purified nucleic acid sequence consisting of at least a portion of a nucleotide sequence selected from the group consisting of (a) SEQ ID NO:1, the complement thereof, variants thereof, and homologs thereof, (b) SEQ ID NO:3, the complement thereof, variants thereof, and homologs thereof, and (c) SEQ ID NO:5, the complement thereof, variants thereof, and homologs thereof. In a preferred embodiment, the nucleic acid sequence encodes at least a portion of the amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof. In a more preferred embodiment, the nucleic acid sequence is double-stranded. In an alternative more preferred embodiment, the nucleic acid sequence is single-stranded.
Without intending to limit the nucleic acid sequences of the invention to any particular type of encoded protein, in an alternative preferred embodiment, the nucleic acid sequence encodes a fusion protein. While it is not contemplated that the invention be limited to the type or nature of fusion partner in the fusion protein, in a more preferred embodiment, the fusion protein comprises a polypeptide selected from the group consisting of chloramphenicol acetyltransferase, luciferase, beta-galactosidase, green fluorescent protein, Myc protein, protein A, glutathione-S-transferase, FLAG tag, and polyhistidine.
In another alternative preferred embodiment, the nucleic acid sequence is contained on a recombinant expression vector. In a more preferred embodiment, the expression vector is contained within a host cell. While it is not contemplated that the invention be limited to the type of host cell, in a yet more preferred embodiment, the host cell is eukaryotic. In an even more preferred embodiment, the eukaryotic cell is selected from the group consisting of cancer cells and amphibian oocytes.
Also provided by the invention is a substantially purified amino acid sequence comprising at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof. In a preferred embodiment, the portion is part of a fusion protein. While not intending to limit the invention to any particular type or nature of fusion protein, in a more preferred embodiment, the fusion protein comprises a polypeptide selected from the group consisting of chloramphenicol acetyltransferase, luciferase, beta-galactosidase, protein A, glutathione-S-transferase, FlAG tag, and polyhistidine.
The invention further provides substantially purified amino acid sequences encoded by at least a portion of a nucleotide sequence selected from the group consisting of (a) SEQ ID NO: 1, the complement thereof, variants thereof, and homologs thereof, (b) SEQ ID NO:3, the complement thereof, variants thereof, and homologs thereof, and (c) SEQ ID NO:5, the complement thereof, variants thereof, and homologs thereof.
The invention additionally provides methods for detecting presence of a nucleic acid sequence encoding at least a portion of a calcium channel protein, comprising: a) providing: i) a sample suspected of containing the nucleic acid sequence; and ii) at least a portion of a nucleotide sequence selected from the group consisting of (1) SEQ ID NO: 1, the complement thereof, variants thereof, and homologs thereof, (2) SEQ ID NO:3, the complement thereof, variants thereof, and homologs thereof, and (3) SEQ ID NO:5, the complement thereof, variants thereof, and homologs thereof, b) combining the sample with at least a portion of the nucleotide sequence under conditions such that the nucleic acid hybridizes with at least a portion of the nucleotide sequence; and c) detecting the hybridization. Although it is not contemplated that the invention be limited to the level of stringency of hybridization, in one preferred embodiment, the hybridization is under conditions of low stringency. In another preferred embodiment, the hybridization is under conditions of high stringency. Furthermore, it is contemplated that the invention will also be used in various assays to detect mRNA using these sequences (i.e., SEQ ID NOS:1, 3, and 5) including, but not limited to microassays.
The invention also provides methods for producing at least a portion of a calcium channel protein, comprising: a) providing: i) a recombinant expression vector comprising a nucleic acid sequence encoding at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof, and ii) a host cell; and b) introducing the vector into the host cell under conditions such that the host cell expresses the portion of the amino acid sequence. In one preferred embodiment, the method further comprises step c) recovering the expressed amino acid sequence (or portion thereof). While not intending to limit the invention to the type or nature of host cell, in an alternative preferred embodiment, the host cell is eukaryotic. In a more preferred embodiment, the eukaryotic host cell is selected from the group consisting of cancer cells and amphibian oocytes.
The invention also provides methods for screening test compounds for modulating calcium channel activity, comprising: a) providing: i) the test compound; ii) a calcium channel selective ion; iii) a control cell; and iv) a host cell comprising a cell membrane and expressing heterologous nucleic acid sequences encoding: 1) calcium channel xcex11 subunit; and 2) at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof; b) contacting the host cell with the test compound and with the molecule to produce a treated host cell; c) depolarizing the cell membrane of the treated host cell under conditions such that the molecule enters the cell through a functional calcium channel; and d) detecting a difference between current flowing into the treated host cell and current flowing into a control cell, thereby identifying the test compound as a compound capable of modulating calcium channel activity. In one preferred embodiment, the method further comprises, prior to the depolarizing, maintaining the treated host cell at a holding potential that substantially inactivates endogenous calcium channels. In another preferred embodiment, the method further comprises, prior to or simultaneously with the step of contacting the host cell with the test compound, contacting the host cell with a calcium channel agonist, wherein the test compound is tested for activity as an antagonist. In yet another preferred embodiment, the host cell further expresses calcium channel beta (xcex2) subunit. In another preferred embodiment, the host cell further expresses calcium channel beta (xcex2) subunit and gamma (xcex3) subunit. Without intending to limit the type or source of host cell, in yet another preferred embodiment, the host cell is eukaryotic. In a more preferred embodiment, the eukaryotic host cell is selected from the group consisting of cancer cells and amphibian oocytes.
Also provided herein is a method of generating antibodies directed against at least a portion of a calcium channel protein, comprising: a) providing: i) at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof; and ii) a host; b) immunizing the host with at least a portion of the amino acid sequence so as to generate an antibody; and c) collecting the antibody from the host. In one preferred embodiment, the method further comprises step d) purifying the antibody. Without limiting the type of host cell, in another preferred embodiment, the host is a mammal. In yet another preferred embodiment, the mammal is a mouse.
The invention further provides an antibody raised according to the method described in the preceding paragraph, i.e., by a) providing: i) at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof; and ii) a host; b) immunizing the host with at least a portion of the amino acid sequence so as to generate antibody; and c) collecting the antibody from the host. In one preferred embodiment, the antibody is monoclonal. In another preferred embodiment, the antibody is polyclonal. In still further embodiments, the present invention provides methods and compositions for genetic immunization for the production of antibodies (e.g., by injecting expression plasmid into an animal).
Also provided herein is an antibody which specifically binds to an amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof. In a preferred embodiment, the antibody is monoclonal. In another preferred embodiment, the antibody is polyclonal.
The invention also provides a method of detecting expression of at least a portion of a calcium channel protein, comprising: a) providing: i) a sample suspected of expressing the calcium channel protein; and ii) an antibody raised according to the above-described method (i.e., by a) providing: i) at least a portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2 and variants thereof, SEQ ID NO:4 and variants thereof, and SEQ ID NO:6 and variants thereof; and ii) a host; b) immunizing the host with at least a portion of the amino acid sequence so as to generate an antibody; and c) collecting the antibody from the host); b) combining the sample and the antibody under conditions such that the antibody binds to the calcium channel protein; and c) detecting the binding between the antibody and the calcium channel protein in the sample.
The present invention additionally provides methods for producing transgenic non-human animals, wherein the animal expresses a reduced level of calcium channel xcex12xcex4 subunit relative to a corresponding wild-type animal, comprising: a) providing: i) an embryonic stem cell comprising wild-type calcium channel xcex12xcex4-subunit genes; ii) a blastocyst of a non-human animal; iii) a pseudopregnant non-human animal; and iv) an oligonucleotide sequence comprising at least a portion of a non-human nucleotide sequence homologous to a nucleic acid sequence selected from the group consisting of (1) SEQ ID NO:1, complements thereof and variants thereof, (2) SEQ ID NO:3, complements thereof and variants thereof, and (3) SEQ ID NO:5, complements thereof and variants thereof; b) introducing the oligonucleotide sequence into the embryonic stem cell under conditions such that the oligonucleotide sequence is homologously recombined into at least one of the wild-type calcium channel xcex12xcex4-subunit genes in the genome of the embryonic stem cell to produce a treated embryonic stem cell; c) injecting the treated embryonic stem cell into the blastocyst to produce an injected blastocyst; d) introducing the injected blastocyst into the pseudopregnant non-human animal; and e) permitting the pseudopregnant animal to deliver progeny comprising the homologously recombined oligonucleotide, wherein the progeny express a reduced level of calcium channel xcex12xcex4-subunit relative to a corresponding wild-type animal. In one preferred embodiment, the transgenic non-human animal is selected from the order Rodentia. In a more preferred embodiment, the transgenic non-human animal is a mouse.
The invention also provides methods for producing transgenic non-human animals, wherein the animal expresses reduced activity of calcium channel xcex12xcex4 subunit relative to a corresponding wild-type animal, comprising: a) providing: i) an embryonic stem cell comprising wild-type calcium channel xcex12xcex4-subunit genes; ii) a blastocyst of a non-human animal; iii) a pseudopregnant non-human animal; and iv) an oligonucleotide sequence comprising at least a portion of a non-human nucleotide sequence homologous to a nucleic acid sequence selected from the group consisting of (1) SEQ ID NO:1, complements thereof and variants thereof, (2) SEQ ID NO:3, complement thereof and variants thereof, and (3) SEQ ID NO:5, complements thereof and variants thereof, wherein at least portion of the nucleotide sequence comprises one or more mutations selected from the group consisting of deletion, insertion and point mutation(s); b) introducing the oligonucleotide sequence into the embryonic stem cell under conditions such that the oligonucleotide sequence is homologously recombined into at least one of the wild-type calcium channel xcex12xcex4-subunit genes in the genome of the embryonic stem cell to produce a treated embryonic stem cell; c) injecting the treated embryonic stem cell into the blastocyst to produce an injected blastocyst; d) introducing the injected blastocyst into the pseudopregnant non-human animal; and e) permitting the pseudopregnant animal to deliver progeny comprising the homologously recombined oligonucleotide, wherein the progeny express reduced activity of calcium channel xcex12xcex4-subunit relative to a corresponding wild-type animal. In one preferred embodiment, the transgenic non-human animal is selected from the order Rodentia. In a more preferred embodiment, the transgenic non-human animal is a mouse.
Additionally provided herein is a method for identifying a therapeutic compound, comprising: a) providing: i) a transgenic non-human animal produced by the method described in either of the previous two paragraphs; and ii) a composition comprising the compound; and b) administering the compound to the transgenic non-human animal to produce a treated animal. While it is not intended that the transgenic animal be limited to a particular type, in one preferred embodiment, the transgenic animal has cancer. In a more preferred embodiment, the cancer is selected from the group consisting of lung cancer, breast cancer, nasopharyngeal cancer, cervical cancer, head cancer and neck cancer. In yet a more preferred embodiment, the lung cancer is selected from the group consisting of small cell carcinoma and non small cell carcinoma. In another preferred embodiment, the transgenic animal has a neurological disease selected from the group consisting of epilepsy, stroke, brain trauma, Alzheimer""s disease, multiinfarct dementia, amyotrophic lateral sclerosis, convulsions, seizures, Huntington""s disease, and amnesia. In yet another preferred embodiment, the transgenic animal has a cardiovascular disease selected from the group consisting of cardiac arrhythmia, angina pectoris, hypoxic damage to the cardiovascular system, ischemic damage to the cardiovascular system, myocardial infarction, and congestive heart failure. In a further preferred embodiment, the transgenic animal has Lambert-Eton myasthenic syndrome.
In still further embodiments, the present invention provides methods for treatment of human tumors in non-human animals. For example, it is contemplated that the methods for identifying therapeutic compounds as described herein will find use in the treatment of human tumors in immunodeficient mice (e.g., nude and/or SCID [severe combined immunodeficiency] mice) or other animals.